Among the risks inherent in handling or being transfused with blood, blood proteins, or other blood components is the risk of infection from pathogenic contaminants, including human immunodeficiency virus (HIV), serum hepatitis, cytomegalovirus, Epstein-Barr virus, herpes simplex, infectious mononucleosis, syphilis and malaria. Virucidal methods, including heat, solvent-detergent, and gamma irradiation, have been used to produce non-infectious plasma derivatives, but such methods are generally ineffective or too harsh to be routinely used for effective decontamination of whole blood, red cells and/or platelets. Indeed, any virucidal treatment that damages or introduces harmful or undesirable contaminants into the product is unsuitable for decontamination of a product intended for transfusion into an animal, particularly a human. Because of the critical need for transfusible red blood cells, it is of great importance to develop methods that can be readily used to decontaminate cellular blood components and whole blood without substantially or irreversibly altering or harming them.
Decontamination treatments that inactivate contaminating pathogens but do not harm the cellular fractions of blood are not readily available. Common decontamination treatments include the use of photosensitizers, which, in the presence of oxygen and upon exposure to light that includes wavelengths absorbed by the photosensitizer, inactivate viruses (EP 0 196 515). Typically, such photochemicals are dyes or other compounds that readily absorb UV or visible light in the presence of oxygen. These compounds include merocyanine 540 ("MC 540") (U.S. Pat. No. 4,775,625) and porphyrin derivatives (U.S. Pat. No. 4,878,891), as well as other photosensitizers.
Increased virucidal activity of these compounds is realized when the absorption spectrum of the photosensitizer does not significantly overlap the absorption spectra of pigments present in the blood, such as hemoglobin. In order to minimize cellular damage, it is preferable that the photosensitizer be nontoxic to cellular blood components and selectively bind to a component of the virus either that is not present in red cells or platelets or, if present therein, that is not essential to red cells' or platelets' function. It is also preferable if the photodynamic treatment inactivates both extracellular and intracellular viruses as well as proviruses. It is further preferable that the virucidal activity of the photosensitizer be uninhibited by the presence of plasma proteins, such as coagulation proteins, albumin and the like.
Treatment with known photochemicals, however, frequently does damage cellular blood components. For example, photochemicals such as the porphyrins (U.S. Pat. No. 4,878,891) and MC 540 (U.S. Pat. No. 4,775,625) cause membrane damage in the presence of light and oxygen, which significantly reduces the survivability of the phototreated red cells during storage. Similarly, treatment of red blood cells using phthalocyanine 4 with type 1/type 2 quenchers caused red cell damage even under optimized conditions--about 2% of the cells hemolyze after 21 days of storage (the current FDA guideline for hemolysis is .ltoreq.1% after 6 weeks of storage at 1-6.degree. C.) (Transfusion 35:367-70 (1995)).
Additionally, both MC 540 and porphyrin derivatives apparently bind to blood components, such as albumin (Transfusion 29:42S (1989); Biochim. Biophys. Acta 1235:428-436 (1995)). For example, the effect of MC 540 on platelets and the influence of albumin on MC 540's virucidal activity has been studied. Platelets exhibited a MC 540 dose-dependent decrease in response to thrombin in the absence of light. In the presence of light and MC 540, the platelets aggregated. Albumin, however, prevented aggregation and inhibited the inactivation of viral contaminants by MC 540 plus light. Similarly, because of such competitive inhibition reactions with blood and/or plasma components, other dyes are not suitable for decontaminating blood, cellular blood components, or any blood-derived products containing high plasma concentrations (as plasma concentration increases, the percentage of viral inactivation substantially decreases).
Charged phenothiazin-5-ium dyes such as methylene blue, toluidine blue O, thionine, azure A, azure B, and azure C have been shown to inactivate animal viruses (U.S. Pat. Nos. 4,407,282, 4,402,318, 4,305,390, and 4,181,128). One target for virus inactivation is viral nucleic acids (Abe et al., Photochem. Photobiol. 61:402-409 (1995)). Methylene blue and visible light damage guanine residues of nucleic acids (Simon et al., J Mol. Biol. 4:488-499 (1962)). Methylene blue and white light produce 8-hydroxy-guanine in DNA (Floyd et al., Arch. Biochim. Biophys. 273:106-111 (1989)). Based on this activity, these dyes have been employed for inactivation of extracellular enveloped viruses in blood and blood components (U.S. Pat. No. 5,545,516).
These particular phenothiazin-5-ium dyes, however, have certain drawbacks that limit their usefulness for inactivating pathogens in whole blood or blood components. For example, red cells readily take up or bind such dyes (Sass et al., J. Lab. Clin. Med 73:744-752 (1969)). In addition, photosensitized oxidation of biological membranes is deleterious to membrane structure and function (methylene blue cross-links the membrane protein, spectrin, in erythrocytes exposed to visible light and oxygen) (Girotti, Biochim. Biophys. Acta. 602:45-56 (1980)). Also, methylene blue treated red blood cells have been shown to bind to plasma proteins, such as IgG and albumin (Wagner et al., Transfusion 33:30-36 (1992)). Finally, because of their hydrophilic character, these dyes cannot readily cross the cell membrane of cellular blood components and so are less effective at reducing the intracellular level of active pathogenic contaminants.
No method has therefore proven fully successful for decontaminating whole blood, blood components, or compositions containing concentrated blood components, including high levels of plasma. There remains, however, an acute need for a safe and effective method for reducing the level of active pathogenic contaminants, particularly HIV and hepatitis, in whole blood or blood components without rendering the blood or blood components unsuitable for transfusion.
Additionally, any method employed to decontaminate blood should not adversely affect the survivability of red blood cells during prolonged storage. During storage, human red blood cells undergo morphological and biochemical changes, including: decreases in the intracellular levels of adenosine triphosphate ("ATP")(associated with the fluidity of the cellular membrane, which is essential for the passage of cells through the narrow channels in the spleen and liver), 2,3 diphosphoglycerate ("2,3-DPG") (which is associated with the ability of the hemoglobin in the red cells to deliver oxygen to the tissues), and potassium (which is associated with, inter alia, transport of ions across the cell membrane); changes in cellular morphology, such as spicule formation (which reduces the surface area of the cell and, as a consequence, the ability of the cell to deform on passing through narrow channels); and progressive hemolysis (which causes an overall reduction in the number of viable cells for transfusion). These changes can be monitored to observe the effects of decontamination methods on the survivability of the red blood cells and their suitability for transfusion.
Solutions that prolong the shelf life of red cells are known (U.S. Pat. No. 4,585,735). Typically, such solutions contain citrate, phosphate, glucose, adenine, and other ingredients and function to prolong shelf life by maintaining the levels of ATP and 2,3-DPG in the cells. Solutions that contain a membrane-penetrating salt, such as ammonium acetate, in addition to phosphate, glucose, and adenine, and that are hypotonic with respect to molecules that are unable to penetrate the cell membrane have been shown to maintain the levels of ATP for more than 100 days of refrigeration (U.S. Pat. No. 4,585,735).